Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects 5.4 million Americans, yet there are still no FDA-approved therapies to treat the underlying causes. One AD characteristic is the deposition of amyloid plaques, which are formed by aggregation of ?-amyloid (A?) peptides. According to the amyloid cascade hypothesis, the accumulation of A? peptides in the brain triggers a pathological cascade that causes neurodegeneration and eventually leads to AD. ?-Secretase is an intramembrane protease complex that controls the processing of numerous protein substrates. It is an appealing drug target for AD since it is responsible for the final step of amyloid precursor protein (APP) proteolysis to generate A? peptides. Genetic evidence from mutations in presenilin (?-secretase catalytic subunit) and APP supports the development of ?-secretase inhibitors (GSIs) to treat AD; however, all AD clinical trials of GSIs have failed. Side effects from inhibition of non-APP substrates (such as Notch) in addition to a lack of knowledge of the molecular basis for controlling enzyme activity and specificity have rendered it a challenging target for drug discovery. In order to develop GSIs with improved potency and selectivity, a better understanding of the interactions between ?-secretase and its substrates is needed. Our lab has discovered a substrate inhibitory domain (ASID) within APP that negatively modulates ?-secretase activity by binding to an allosteric site in the enzyme complex. Interestingly, ASID-derived inhibitors preferentially reduce ?-secretase-mediated A? production over Notch cleavage. Such an inhibitory domain within the substrate could represent a common regulatory mechanism in ?-secretase and other enzymes. The objective of this application is to determine how the inhibitory domain of ?-secretase substrates functions to modulate the enzyme. We hypothesize that ?-secretase exists in a constitutively active form and that the substrates themselves serve as key regulators to modulate their own cleavage by ?-secretase via unique inhibitory domains. Disruption of this negative modulation in APP can potentially result in elevated A? production and ultimately lead to AD. Through detailed biochemical characterization, this study aims to: 1) determine how ASID interacts with other regions of APP to regulate ?-secretase and 2) identify a substrate inhibitory domain (ASID) in the Notch1 substrate and characterize its interaction with ?-secretase. In order to investigate the mechanism of action for ASID, we will synthesize peptides and chemical probes to examine the relationship of the ASID site with the docking site and active site of ?-secretase. We will develop a series of Notch1 substrates and inhibitors to validate the existence of a Notch substrate inhibitory domain and elucidate its regulatory mechanism. The proposed studies will decode the molecular mechanism of this novel regulation of ?-secretase through the inhibitory domains located within the substrates. Findings from this proposal will provide critical insight into the processes underlying AD and open a new avenue for development of more effective GSIs.